The present invention relates to a pigment epithelial cell of the eye comprising vector DNA of an adenoviral vector with large DNA capacity, to the improved isolation and cultivation of these cells and to methods for the production and the use in therapy of an eye or nerve disease.
The five primary senses of touch, sight, hearing, taste and smell serve to pick up information from the surroundings. About 75% of our perceptions involve the sense of sight. This high percentage makes it clear that sight is of predominant importance in our daily life. Consequently, a weakening of our vision represents a great intrusion into everyday life.
The eye consists of a compound lens system which produces an image, which is inverted and reduced in size, of the surroundings on the retina. The dioptic apparatus consists of the transparent cornea, the iris forming the pupil, the lens and the vitreous body, a gelatinous, transparent mass inside the eyeball between lens and retina. FIG. 1 shows a schematic horizontal section through the eye. The covering of the eyeball consists of 3 layers: the sclera, the choroid and the retina. The retina in turn consists of an outer layer, the retinal pigment epithelium (RPE), and an inner layer, the neurosensory retina.
The annular iris separates the anterior from the posterior chamber of the eye and forms the anterior part of the uvea. From anterior to posterior it consists of a collagen-rich extracellular matrix, the iris stroma (it contains melanocytes, fibrocytes, nerves and blood vessels) and the iris pigment epithelium (IPE).
The iris pigment epithelium is in two layers and consists of an anterior and a posterior pigment epithelial cell layer (Freddo TF (1996) Ultrastructure of the iris. Microsc Res Tech 33: 369–389). The cells of the posterior iris pigment epithelium are connected by tight junctions. The anterior pigment epithelium has in addition smooth muscle cells (except in the region of the sphincter) which contribute to dilatation of the iris (Freddo 1996, supra). The iris pigment epithelium has the same embryological origin as the retinal pigment epithelium. It is possible to obtain about 2.3×105 IPE cells from a human iris, 90% of which survive in cell culture (Hu DN, Ritch R, McCormick SA, Pelton-Henrion K(1992) Isolation and cultivation of human iris pigment epithelium. Invest Ophthalmol Vis Sci 33: 2443–2453). IPE cells are highly pigmented and contain much eumelanin. Melanin has the following protective functions. It is able to bind divalent iron ions (Fe2+) and other toxic substances (e.g. Ca2+) and thus remove them from the cytoplasm of the cell (Hill HZ (1992) The function of melanin or six blind people examine an elephant. Bioessays 14: 49–56). Melanin is additionally able to convert Fe2+ into less toxic Fe3+ by redox reactions. On the other hand, the melanin synthesis precursors dihydroxyindole (DHI) and dihydroxyindolecarboxylic acid (DHIA) have a very strong antioxidant effect which is stronger than that of alpha-tocopherol (Memoli S, Napolitano A, d'Ischia M, Misuraca G, Palumbo A, Prota G (1997) Diffusible melanin-related metabolites are potent inhibitors of lipid peroxidation. Biochim Biophys Acta 1346: 61–68). Melanin is able to eliminate toxic oxygen free radicals produced in the eye by the high partial pressure of oxygen in combination with exposure to light. Elements important for the normal function of the retina, such as, for example, zinc, are moreover stored by melanin with great efficiency. Zinc, as a cofactor for, for example, antioxidative enzymes (superoxide dismutase) or connective tissue-degrading enzymes (metalloproteinases), has several important functions in the eye and in the central nervous system (CNS).
The pigment epithelium plays an import part in metabolism and in absorption of light in the eye. It is additionally responsible for the outer blood-retina barrier and for disposing of rejected photoreceptor cells. Consequently it forms an interesting target for the gene therapeutic treatment of eye diseases.
To date, a few experiments on the genetic modification of pigment epithelial cells have been described, but these provided unsatisfactory results in terms of the duration and stability of expression.
In a study with laboratory mice, a first generation adenoviral vector which expressed the E.coli lacZ gene under the control of the CMV promoter was used for gene transfer into the retinal pigment epithelium by subretinal injection. First generation adenoviral vectors (Gilardi et al., FEBS Letters 267, 60–62, 1990; Stratford-Perricaudet et al., Hum. Gene Ther. 1, 241–256, 1990) are characterized by deletions of the E1A and E1B genes. E1A and E1B have transforming and transactivating properties. In some vectors there is also deletion of E3 in order to increase the capacity to take up foreign DNA. Although the gene transfer into the retinal pigment epithelium was efficient and very good expression was observed shortly after injection in the retinal pigment epithelium, the expression was transient. 6 weeks after the injection, only a few lacZ-positive retinal pigment epithelial cells were still observable (Li T, Adamian M, Roof DJ, Berson EL, Dryia TP, Roessler BJ, Davidson BL (1994) In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector. Invest Ophthalmol Vis Sci: 35, 2543–2549).
A further study carried out on laboratory rats with an observation period of 14 days used a first generation adenovirus which expressed the E.coli lacZ gene under the control of the Rous Sarcoma Virus (RSV) promoter. Although the gene transfer into the retinal pigment epithelium was efficient, and very good expression was observed 7 days after the injection in the retinal pigment epithelium, the expression was reduced one week later (Rakoczy PE, Lai CM, Shen WY, Daw N, Constable IJ (1998) Recombinant adenovirus-mediated gene delivery into the rat retinal pigment epithelium in vivo. Australian and New Zealand Journal of Ophthalmology 26 (Suppl.): S56–S58).
Another study carried out on 6-week-old RCS rats used a first generation adenoviral vector which expressed the green fluorescence protein (GFP) gene under the control of the CMV promoter (Anglade E, Csaky KG (1998) Recombinant adenovirus-mediated gene transfer into the adult rat retina. Curr Eye Res 17: 316–321). Although the gene transfer into the retinal pigment epithelium after subretinal injection was efficient, and 30 to 90% of the retinal pigment epithelium were GFP-positive in the region of the injection site 3 days after the injection, GFP expression was no longer detectable 6 days later (that is 9 days after the injection).
Whereas first generation adenoviral vectors were used in the above-mentioned examples of gene transfer in the region of the eye, in a further publication there was use of an adenoviral vector which is referred to as adenovirus minichromosome (EAM) for subretinal injection of mice (Kumar-Singh R, Farber DB (1998) Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration. Hum Mol Genet 7: 1893–1900). This comprises a vector which does not express any viral proteins. The vector expressed the beta unit of cyclic GMP phosphodiesterase (PDE) under the control of the natural PDE promoter. The vector also expressed the E.coli lacZ gene under the control of the CMV promoter. In addition, the vector contained various E.coli plasmid elements (Plasmid backbone, ampicillin resistance gene, E.coli origin of replication). After production, the vector was characterized by pronounced variability of its genome. Monomeric and dimeric structures were observed, the latter in head-to-head, head-to-tail and tail-to-tail orientation. Because of this variability and the presence of plasmid sequences including antibiotic resistance, this vector is unsuitable for therapeutic use. The gene transfer experiments were carried out on rd mice which represent an animal model of retinal degeneration and are characterized by a mutation, which causes the degeneration, in the beta unit of the PDE gene. In this study, expression was detected exclusively in the neuronal portion of the retina but not in the retinal pigment epithelium. Although the neuronal cells are post-mitotic and thus no longer able to divide, expression of the PDE gene was only transient. Various methods (RT-PCR, Western blot analysis and determination of the PDE activity) were used to demonstrate that expression was no longer detectable 4 months after the injection.
To date, only the nonviral transfection reagent Lipofectamine has been used for transfecting IPE cells. In this study, the plasmid pXCN2-bFGF which expresses the rat bFGF cDNA was produced. The plasmid additionally contains a neomycin resistance gene. Cultivated rat IPE cells were transfected with this plasmid. The cells expressed the bFGF cDNA in vitro, and the authors write that degeneration of photoreceptors was delayed by up to 4 weeks in the RCS rat after subretinal transplantation as cell suspension (Tamai M, Yamada K, Takeda N, Tomita H, Abe T, Kojima S, Ishiguro I (1997) bFGF transfected iris pigment epithelial cells rescue photoreceptor cell degeneration in RCS rats. In: La Vail M, eds. Degenerative retinal diseases. 323–328). However, since, as shown in the work mentioned, the same effect, namely delayed degeneration of photoreceptors, was observed also in rats which had received IPE cells after transfection with a control plasmid by subretinal injection, and was not improved or extended by the transfection, this effect was not one which could be attributed to a targeted gene transfer but was explicable solely by the transplantation of the IPE cells. In addition, bFGF expression after transplantation was not demonstrated.
The publications mentioned therefore do not disclose an expression system for pigment epithelial cells of the eye with which long-term stable expression of an introduced gene can be observed. Long-term stable expression of such a gene is, however, necessary for the therapy of a large number of hereditary and acquired eye diseases. For many applications it is precisely the long-term production of therapeutic proteins which is the crucial factor for achieving a therapeutic effect.